Multiplexed MRM-Based Proteomics Identified Multiple Biomarkers of Disease Severity in Human Heart Failure

Abstract

Heart failure (HF) is a complex disease due to the intricate interplay of several mechanisms, which therefore implies the need for a multimarker strategy to better personalize the care of patients with HF. In this study, we developed a targeted mass spectrometry approach based on multiple reaction monitoring (MRM) to measure multiple circulating protein biomarkers, involved in cardiovascular disease, to address their relevance in the human HF, intending to assess the feasibility of the workflow in the disease monitoring and risk stratification. In this study, we analyzed a total of 60 plasma proteins in 30 plasma samples from eight control subjects and 22 age- and gender- matched HF patients. We identified a panel of four plasma proteins, namely Neuropilin-2, Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were more abundant in HF patients in relation to disease severity and pulmonary dysfunction. Moreover, we showed the ability of the combination of these candidate proteins to discriminate, with sufficient accuracy, HF patients from healthy subjects. In conclusion, we demonstrated the feasibility and potential of a proteomic workflow based on MRM mass spectrometry for the evaluation of multiple proteins in human plasma and the identification of a panel of biomarkers of HF severity.

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“…This in turn enables the comparison of biomarkers with large abundance differences within one run, thus facilitating multiplexing of many biomarkers in parallel, even without the need for matrix depletion [45]. Given the simplicity, flexibility, and multiplexability of MRM-based LC-MS/MS assays, the initial panel of peptides can be large, increasing the chance of finding a reliable set of reproducible biomarkers based on hierarchical filtering and selection of the most suitable candidates during establishment of the targeted assay [46,47]. Finally, although LC-MRM assays require analytical proficiency and are labor-intensive to set up, they are highly cost-effective to run and can be adapted directly by routine clinical laboratory scientists to meet a change in needs.…”

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“…The plasma was then divided into aliquots, and frozen at -80 °C until analysis. Plasma samples were digested using the ProteinWorks™ eXpress Digest kit (Waters Corporation, Milford, MA, USA) according to the manufacturer’s instructions and as previously described [ 8 ]. Briefly, 35 μL of plasma were mixed with 12 μL of 200 μg/mL Intact mAb Mass Check Standard (Waters Corporation), used as an internal LC-MS standard for MS optimization and performance testing, and then diluted to a total volume of 120 μL using the digestion buffer provided in the kit.…”

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“…Nrp2 deficiency leads to increased vascular permeability with resultant edema [ 48 ]. Nrp2 was one of four plasma proteins, along with Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were increased in heart failure patients in relation to disease severity and pulmonary dysfunction [ 152 ]. Additionally, plasma Nrp2 levels were predictive of heart failure rehospitalizations in patients with preserved ejection fraction, and all-cause mortality in patients with reduced or preserved ejection fraction [ 153 ].…”